Research Paper Volume 13, Issue 16 pp 20302—20318

Regulation of follistatin-like 3 expression by miR-486-5p modulates gastric cancer cell proliferation, migration and tumor progression

Evaluating miR-486-5p specificity for FSTL3 mRNA. (A) Western blotting evaluation of FSTL3 protein levels after transfection of sh-FSTL3 and/or miR-486-5p inhibitor into MGC-803 gastric cancer cells. Monitoring β-actin levels was used as an internal control in these blots. (B) Western blot results were quantified by using Image J software. Error bars indicate +SEM; significance indicated by asterisks, **PPC) Transfection of sh-FSTL3 and/or miR-486-5p inhibitor into MGC-803 gastric cancer cells and evaluation of cell proliferation using EdU assay (24h). (D) Wounded cell monolayer closure assay after transfection of sh-FSTL3 and/or miR-486-5p inhibitor followed by imaging at 8 and 24 h post-wounding. (E) The Transwell assay for cell migration after transfection of sh-FSTL3 and/or miR-486-5p inhibitor was into MGC-803 cells. Sh-NC construct (shRNA) is a negative control for FSTL3 knockdown. (F) Quantified results of EdU, wound healing and transwell migration assays. Sh-NC was analyzed as a control.

Figure 6. Evaluating miR-486-5p specificity for FSTL3 mRNA. (A) Western blotting evaluation of FSTL3 protein levels after transfection of sh-FSTL3 and/or miR-486-5p inhibitor into MGC-803 gastric cancer cells. Monitoring β-actin levels was used as an internal control in these blots. (B) Western blot results were quantified by using Image J software. Error bars indicate +SEM; significance indicated by asterisks, **P<0.01, ***P<0.001; number of experiments, n=3. (C) Transfection of sh-FSTL3 and/or miR-486-5p inhibitor into MGC-803 gastric cancer cells and evaluation of cell proliferation using EdU assay (24h). (D) Wounded cell monolayer closure assay after transfection of sh-FSTL3 and/or miR-486-5p inhibitor followed by imaging at 8 and 24 h post-wounding. (E) The Transwell assay for cell migration after transfection of sh-FSTL3 and/or miR-486-5p inhibitor was into MGC-803 cells. Sh-NC construct (shRNA) is a negative control for FSTL3 knockdown. (F) Quantified results of EdU, wound healing and transwell migration assays. Sh-NC was analyzed as a control.